Despite advances in the therapy of opportunistic infections complicating AIDS, they remain a leading cause of morbidity and mortality. New agents are needed to combat infections by Pneumocystis carinii, Toxoplasma gondii and Mycobacterium tuberculosis and avium. We are in the process of cloning, expressing and characterizing three enzymes from these organisms to provide in vitro targets for the development of new drugs; these enzymes are dihydrofolate reductase (DHFR), thymidylate synthase (TS), and dihydropteroate synthase (DHPS). We will compare the interactions of inhibitors with P. carinii and human DHFR using binding studies and site directed mutagenesis guided by molecular modeling. The objective is to identify the molecular interactions which lead to species discrimination of inhibition. We will also prepare 15N-13C labeled Pneumocystis DHFR for NMR structural studies in collaboration with a collaborator. Toxoplasma TS-DHFR will be expressed, purified and characterized, and the RS and DHFR domains will be separated by manipulation of the gene to facilitate structural characterization. Mycobacterial TS and DHFR clones will be sequenced and the enzymes will be expressed in E. coli. Clones encoding DHPS from Toxoplasma and Mycobacterium will be isolated from cDNA and genomic DNA libraries. These and the Pneumocystis DHPS will be expressed, purified and characterized. Expression of enzymes will be achieved by subcloning coding sequences into E. coli or yeast expression systems. The enzymes will be purified using specific affinity systems when available or by combinations of conventional chromatography. DHFRs will be screened with existing inhibitors. We have established collaborations for x-ray crystallographic structural determinations. The availability of in vitro enzyme systems is providing a valuable resource for the identification and design of new inhibitors of de novo thymidylate synthesis and folate metabolism.
